Poster at the 40th annual meeting of the Biopysical Society . Abstract: ,~ Biophys. J. 70, A335 Though the molecular steps are becoming elucidated, the physical mechanism of cytokinesis is not yet understood. We report here studies of dividing RBL-2H3 cells using two-photon excited fluorescence microscopy. This technique enables three-dimensional resolution, access to a wide spectrum of dyes, and the opportunity for extended imaging on viable specimens. In dividing cells, located in the adhered culture using a vital DNA stain, several acyl-labeled, saturated plasma membrane probes exhibit drastic fluorescence enhancements that are localized to the cleavage furrow and coincident with furrow invagination. These order of magnitude fluorescence increases persist until cell division is complete. We obtain optical, three-dimensional images of mid-body structures that resemble those of electron dense material in TEM micrographs. Using fluorescent lipids of various types we are currently characterizing the physical properties of these distinctive regions. Whether these fluorescence enhancements are due to a complex folding that increases membrane volume or to a segregation of the lipid probes to specialized membrane domains is not yet known.